Structure of fibroblastic intermediate filaments: analysis of scanning transmission electron microscopy.

Abstract

The structure of fibroblastic intermediate filaments from Chinese hamster ovary cells has been investigated by scanning transmission electron microscopy. Freshly extracted (native) filaments were compared with filaments reassembled in vitro from purified decamin. From digital micrographs of unstained specimens, direct measurements of linear mass density were performed on many individual filaments. Native filaments beyond a certain minimal length constitute a homogeneous population, averaging 38 ± 4 kilodaltons (kDal)/nm. A minor but distinct polymorphic variant (23 ± 4 kDal/nm) was also present as very short filaments or end-segments; these may represent breakdown products or assembly intermediates. Analysis of reassembled filaments demonstrates that the in vitro assembly reaction is-in the main-faithful, although the distribution of their mass measurements is appreciably broader than that of the native data. In addition to the predominant type at 37 ± 4 kDal/nm and a minor component at 26 ± 4 kDal/nm, small amounts of a third, more massive, polymorphic variant at 52 ± 5 kDal/nm were also present. Micrographs of negatively stained specimens clearly demonstrate that the filaments are composed of bundles of protofilaments-each 2-3 nm in diameter-and also reveal an axial periodicity of about 46 nm. The implications of these findings are discussed for three classes of model previously proposed for the structure of intermediate filaments.Reassembly in vitro of supramolecular structures from purified constituents is an experimental approach that has contributed greatly to the understanding of how such entities are formed and regulated in the living cell. In such cases as viruses or multimeric enzymes, the success of a reassembly experiment may be demonstrated by a functional assay based on infectivity or catalytic activity. However, with systems whose functional properties are either lacking or undefined, evidence for faithful reassembly is based primarily on structural criteria determined by physical techniques such as electron microscopy and x-ray diffraction.Intermediate filaments (IF) are long fibrous protein polymers that are ubiquitous cytoskeletal constituents of eukaryotic cells (1-3). Five major subclasses ofIF have been distinguished (4), their proteins have been isolated and characterized, and conditions under which they may be induced to self-assemble in vitro have been determined (5). The principal criterion of faithful reassembly has been morphology as defined by electron microscopy. For certain subclasses of native and reassembled IF, x-ray diffraction has also been used to demonstrate the presence ofcoiled-coil a-helices in approximate alignment with the filament axis (6-11). However, quantitative correspondence of a-helical content has not been established and the mode of packing of protein subunits in either reassembled or native IF has yet to be determined.To obtain more rigorous structural information, we have examined fibroblastic IF by scanning transmission electron microscopy (STEM). Bo...

Helical structure of Bordetella pertussis fimbriae

“…Bacteria were collected by centrifugation for 30 min at 5,000 x g. The bacterial pellet was suspended to 20% (wt/vol) in 0.125 M ethanolamine (pH 10.5) and blended in a Sorvall Omni-mixer as described previously (26 (23). After adsorption to a thin carbon film substrate, the fimbriae were washed and freeze-dried as described previously (9,20 recorded directly in digital form with sampling by the focused electron probe (diameter, -0.25 nm) at intervals of 0.5 or 1.0 nm. Mass-per-unit-length analyses were performed as described previously (21), working with the dark-field signal recorded by the high-angle annular detector (9,20).…”

“…After adsorption to a thin carbon film substrate, the fimbriae were washed and freeze-dried as described previously (9,20 recorded directly in digital form with sampling by the focused electron probe (diameter, -0.25 nm) at intervals of 0.5 or 1.0 nm. Mass-per-unit-length analyses were performed as described previously (21), working with the dark-field signal recorded by the high-angle annular detector (9,20). Similarly, radial reconstructions (17) were done with the PIC program (22) implemented on a VAX 11/780 computer and accompanying image-processing hardware.…”

Molecular Parameters of Type IV α-Internexin and Type IV-Type III α-Internexin-Vimentin Copolymer Intermediate Filaments

“…Determination of the exact alignments of the A 11 and A 22 modes has revealed a fourth mode termed A CN , in which the end of one molecule is overlapped by about 1 nm with the beginning of the next parallel molecule in the same axial row. Third, quantitative mass measurements suggest that 12-24 molecules, most commonly 16 in native IF (13)(14)(15), associate in three dimensions in as yet unknown ways to form an IF.…”

Functional changes of intermediate filaments in fibroblastic cells revealed by a monoclonal antibody.

“…In fibroblastic cells, the main constituent of the filaments is vimentin (2, 3), with a molecular weight of 58,000; smaller related peptides (molecular weight, >40,000) are also observed (4, 5) and are thought to be cleavage products of vimentin by a specific protease (6). Vimentin molecules associate to form protofilaments: various models have been proposed for this association (7,8). Protofilaments are associated in bundles; in muscle cells and nucleated erythrocytes, they are crosslinked by another protein, synemin (9, 10); protofilaments are aggregated by cationic proteins (11).…”